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A surplus of nitric oxide (NO) generally spells bad news for neurons, as does mitochondrial dysfunction. Researchers have now discovered a molecular mechanism that links the two within the context of Aβ-induced neurodegeneration. As reported today in Science, that link is dynamin-related protein 1 (Drp1). This protein normally functions to regulate mitochondrial fission, but seems to get thrown out of whack by NO that is made in response to Aβ. The NO induces S-nitrosylation of Drp1, which leads to excess mitochondrial fragmentation that triggers collapse of dendritic spines and eventual neuronal death. Remarkably, a single cysteine mutation in Drp1 that prevents S-nitrosylation appears to protect neurons from Aβ-induced neurotoxicity. “The bottom line here is that [the study] shows on a molecular level how mitochondria are involved in Alzheimer disease, and it provides a new target for therapy,” said lead investigator Stuart Lipton of the Burnham Institute for Medical Research in La Jolla, California.

Wayward mitochondria have been linked to Parkinson (see ARF related news story) and Huntington diseases (see ARF related news story) in addition to AD (see ARF related news story). Several years ago, Lipton’s group, with Ella Bossy-Wetzel of the Burnham Institute, showed that NO in primary cortical neuron cultures sent mitochondria into a fission frenzy, which preceded neurite injury and cell death (Barsoum et al., 2006). The profound fission seemed to jibe with an electron microscopy study that documented fragmented, unusually small mitochondria in the brains of AD patients (Baloyannis, 2006). Since the fission-inducing mitochondrial protein Drp1 is regulated by S-nitrosylation, first author Dong-Hyung Cho and colleagues wondered whether NO exerted its effects on mitochondria by inducing this particular post-translational modification on Drp1.

To test their hunch, the scientists exposed cerebrocortical neurons to the NO donor S-nitrosocysteine (SNOC) and looked to see whether it reacted with Drp1. It did, forming SNO-Drp1 in neurons before triggering mitochondrial fission and eventually fragmentation. The researchers then showed that Drp1 could be S-nitrosylated by endogenous NO and that they could block this reaction with an NOS inhibitor (N-nitro-L-arginine, or NNA).

Building the case for AD relevance of this reaction, Lipton’s team looked for SNO-Drp1 in Aβ-treated neuronal cultures under conditions that typically induce mitochondrial dysfunction and subsequent neuronal damage. Sure enough, SNO-Drp1 appeared when the neurons were exposed to Aβ25-35 oligomers, or to endogenously produced Aβ from conditioned medium of APP-overexpressing cell lines. Furthermore, the scientists found elevated levels of SNO-Drp1 in APP-overexpressing AD transgenic mice (Tg2576) and in postmortem brain tissue from AD patients, but not in brains of those who died of PD or non-CNS causes.

Using mutational analysis, the team found that S-nitrosylation of Drp1 required but a single cysteine (Cys644) in the protein’s GTPase domain. The researchers went on to show that S-nitrosylation of this residue enhanced dimerization of purified Drp1 proteins and increased their GTPase activity, whereas these effects disappeared when Drp1(C644A) mutant proteins were tested. In a similar manner, transfection of cortical neurons with wild-type Drp1 promoted mitochondrial fragmentation, loss of dendritic spines, and cell death in response to NO, and expression of the C644A mutant rescued neurons from these problems.

“Overall, the work supports the notion that abnormal mitochondrial dynamics play a critical role in mitochondrial dysfunction and neuronal injury in AD, and it sheds new light on the mechanisms underlying mitochondrial dysfunction and its relation to synaptic dysfunction,” Xiongwei Zhu of Case Western Reserve University in Cleveland, Ohio, wrote in an e-mail to ARF (see full comment below). In a paper published last fall, Zhu’s group showed that Aβ overproduction caused mitochondria to break apart and distribute abnormally in neurons, and that these anomalies contribute to neuronal dysfunction (Wang et al., 2008).

Moving forward with the new findings, Lipton and colleagues have begun a high-throughput screen for drugs that block the S-nitrosylation reaction at Cys644 on Drp1. This target is “totally different” from those of other AD drug candidates, Lipton said.—Esther Landhuis

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To explore the mechanisms underlying nitric oxide- and Aβ-induced mitochondrial fragmentation in neurons (Barsoum et al., 2006), Stuart Lipton’s group convincingly demonstrates that NO induces S-nitrosylation of Drp1 at Cys644 that enhances Drp1 dimerization and increases GTPase activity, thus leading to excessive mitochondrial fission. This study adds one additional layer, in addition to phosphorylation, sumoylation and ubiquitination, to the post-transcriptional modification and regulation of Drp1. The presence of S-nitrosylated Drp1 in Aβ25-35 treated neurons implicates a similar mechanism that likely contributes to Aβ-induced mitochondrial fragmentation. Indeed, that Drp1(C644A) expression abrogates the adverse effects of naturally secreted Aβ on spine density indirectly supports such a notion.

Most importantly, the presence of S-nitrosylated Drp1 in Tg2576 and AD brain tissues lends pathophysiological significance to this mechanism in vivo. Overall, the work presented supports the notion that abnormal mitochondrial dynamics play a critical role in mitochondrial dysfunction and neuronal injury in AD brain, and it sheds new light on the mechanisms underlying mitochondrial dysfunction and its relation to synaptic dysfunction. Given that abnormal mitochondrial fission and fusion are increasingly implicated in other neurodegenerative diseases such as Parkinson's and Huntington's (Frank et al., 2001; Lee et al., 2004; Chen et al., 2005; McBride et al., 2006; Parone et al., 2006; Yu et al., 2006; Lee et al., 2007), it would be of interest to see whether similar mechanisms also play a role in those diseases.

Both mitochondrial dysfunction and synaptic dysfunction are early events during AD pathogenesis; however, whether and how the former affects the latter was unclear. Recently we demonstrated that Aβ overproduction causes both mitochondrial fragmentation and abnormal distribution in neuronal cells (Wang et al., 2008). We also demonstrated that excessive fragmentation compromised mitochondrial function. This, together with an abnormal distribution, likely contributes to synaptic dysfunction. Although we demonstrated that an imbalance in the mitochondrial fission and fusion machinery was directly responsible for Aβ-induced mitochondrial fragmentation, the present study offers another mechanism through enhanced S-nitrosylation of Drp1. In vivo, it is likely that both mechanisms may be in play.

Nonetheless, since enhanced mitochondrial fission does not necessarily cause synaptic dysfunction (Li et al., 2004), it remains to be determined whether and how Drp1(C644A) affects mitochondrial function and distribution and thus abrogated the adverse effect of naturally secreted Aβ on spine density.